This experiment was to evaluate the effect of dietary resveratrol (Res) supplementation (0, 400 mg/kg) on growth performance, meat quality, and muscle anaerobic glycolysis and antioxidant capacity of transported broilers. A total of 360 21-day-old male Cobb broilers was randomly allotted to 2 dietary treatments (Res-free group and Res group) with 12 replicates of 15 birds each. On the morning of d 42, after a 9-hour fast, 24 birds (2 birds of each replicate) were selected from the Res-free group and then equally placed into 2 crates, and the other 12 birds (one bird of each replicate) were selected from the Res group and then placed into the other crate. All birds in the 3 crates were transported according to the following protocols: 0-hour transport of birds in the Res-free group (control group), 3-hour transport of birds in the Res-free group (T group), and 3-hour transport of birds in the Res group (T + Res group). The results showed that Res not only improved feed conversion ratio (P < 0.05) but also tended to improve birds’ final body weight (P < 0.10). In the Res-free group, a 3-hour transport increased serum corticosterone concentration, muscle malondialdehyde (MDA) and lactate contents, and muscle lactate dehydrogenase (LDH) activity, while it decreased muscle glycogen content, total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) activities (P < 0.05), which induced decreased breast meat quality (lower pH24h and higher drip loss and L*24 h, P < 0.05). Nevertheless, compared with the T group, Res increased muscle glycogen content and T-SOD and GSH-PX activities (P < 0.05 or P < 0.10), while it decreased muscle MDA content and LDH activity (P < 0.05), which is beneficial to the meat quality maintenance of transported broilers (lower drip loss, L*24 h, and higher pH24h, P < 0.05 or P < 0.10). This study provides the first evidence that dietary resveratrol supplementation prevents transport-stress-impaired meat quality of broilers, possibly through decreasing the muscle anaerobic glycolysis metabolism and improving the muscle antioxidant capacity.
Lipases are ubiquitous enzymes in nature, which play a crucial role in fat metabolism by catalyzing the hydrolysis of triacylglycerol to free fatty acids and glycerol. However, reports concerning insect lipase are rare. In this study, we studied the expression and activity of a lipase-related protein from Antheraea pernyi (ApLRP). Recombinant ApLRP was expressed in Escherichia coli cells and used to raise rabbit anti-ApLRP polyclonal antibodies. ApLRP mRNA and protein expression were abundant in the midgut and malpighian tubules, respectively. After challenge with four different microorganisms (E. coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus), the expression levels of ApLRP mRNA in midgut were inducted significantly compared with the control. The different pathogens induced different ApLRP gene expression patterns. The optimum temperature and pH for the enzyme's activity were 35°C and 7.0, respectively. ApLRP activity was stimulated in the presence of Mg2+, Na+, Ca2+ and b-mercaptoethanol; while Zn2+, Cu2+ and Fe3+ inhibited its activity. Detergents such as SDS, glycerol and Tween-20 increased the lipase activity by 20-30%. Our results indicated that ApLRP might play an important role in the innate immunity of insects.
The E2F transcription factor family is distributed widely in eukaryotes and has been well studied among mammals. In the present study, the E2F transcription factor 4 (E2F4) gene was isolated from fat bodies of Antheraea pernyi and sequenced. E2F4 comprised a 795 bp open reading frame encoding a deduced amino acid sequence of 264 amino acid residues. The recombinant protein was expressed in Escherichia coli (Transetta DE3), and anti-E2F4 antibodies were prepared. The deduced amino acid sequence displayed significant homology to an E2F4-like protein from Bombyx mori L. Quantitative real-time polymerase chain reaction analysis revealed that E2F4 expression was highest in the integument, followed by the fat body, silk glands, and haemocytes. The expression of E2F4 was upregulated in larvae challenged by bacterial (Escherichia coli, Micrococcus luteus), viral (nuclear polyhedrosis virus), and fungal (Beauveria bassiana) pathogens. These observations indicated that E2F4 is an inducible protein in the immune response of A. pernyi and probably in other insects.
Serpins are a broadly distributed family of proteases found in various organisms that play an important role in regulating the immune response. Here, we identified a serpin-1 gene from Antheraea pernyi that encodes a 279 amino acid protein with a molecular weight of 30.8 kDa. We expressed the recombinant Ap-serpin-1 protein in Escherichia coli and used the purified protein to prepare rabbit anti-Ap-serpin-1 polyclonal antibodies. We calculated the enzyme-linked immunosorbent assay titer of the antibody as 1:128000. Quantitative real-time polymerase chain reaction analysis revealed that Ap-serpin-1 was expressed in all examined tissues, including hemolymph, malpighian tubules, midgut, silk gland, integument and the fat body; the highest Ap-serpin-1 expression levels was detected in the fat body. We next investigated the expression patterns of Ap-serpin-1 in both fat body and hemolymph samples, following treatment with E. coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus (NPV). We reported that NPV and M. luteus significantly enhanced Ap-serpin-1 expression in the fat body. While, in the hemolymph samples, treatment with B. bassiana and M. luteus was shown to upregulate Ap-serpin-1 expression at 24 h induction. Altogether, our results suggest that Ap-serpin-1 is involved in the innate immunity of A. pernyi.
The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock‐down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far‐western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP‐1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase‐activating system, and it may play an important role in regulation of the innate immunity in insects.
ABSTRACT. Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Grampositive bacteria, Gram-negative bacteria, viruses, and fungi using real- time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point.
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